Characterization of Bioactive Compounds from Romanian

Cetraria islandica (L) Ach.

SIMONA PATRICHE1, IOANA OTILIA GHINEA1, GIGI ADAM2, GABRIELA GURAU2, BIANCA FURDUI1, RODICA MIHAELA DINICA1*,
LAURA-FLORENTINA REBEGEA2, MARIANA LUPOAE2
1
Dunarea de Jos University of Galati, Faculty of Science and Environment, Chemistry, Physics and Environment Department,
47 Domneasca Str., 800008, Galati, Romania
2
Dunarea de Jos University Galati, Faculty of Medicine and Pharmacy, 35 Al. I. Cuza Str, 800010, Galati, Romania

Lichens (Lichenophyta phylum), the least exploited subdivision of fungus, are composite plants used in folk
medicine for the treatment of diverse pathologies, from respiratory to digestive diseases, as they contain
over 500 potentially bioactive compounds identified up-to-date. Lichen acids, such as usnic acid, lobar acid,
lecanoric acid or salazinic acid, are among these compounds, with biopharmaceutical applications as
antimicrobial, antioxidant and cytotoxic agents. The objectives of this study were to extract usnic acid from
Romanian Cetraria islandica, (Parmeliaceae family), to characterize the extracts and to evaluate their
antioxidant and antimicrobial potential. The extracts were characterized using FTIR spectroscopy and
HPTLC techniques. The extracts and pure usnic acid have shown high antioxidant activity and have activity
against certain Gram-positive and Gram-negative bacteria and fungi such as Candida albicans. Therefore,
the analyzed bioactive compounds could be used as the basis of pharmaceutical formulations to treat
various respiratory and digestive disorders.
Keywords: lichens, usnic acid, IR spectroscopy, HPTLC, antioxidant, antimicrobial

Lichens are complex symbiotic associations between
a fungus (mycobiont) and an alga (photobiont) used in
traditional medicine for various diseases due to the
presence of several bioactive compounds in their structure
[1]. Cetraria islandica lichen is one of the most used species
to treat tuberculosis, acute respiratory diseases, especially
those of upper tract, inflammation of the throat and oral
cavity, stomach and duodenal ulcers [2]. Furthermore,
Cetraria islandica has a significant role to reduce the acidity
and adjust the digestion and the absorption after severe
illnesses or surgeries [3]. 700 compounds are found in
lichen structure, of which about 200 are depside and over
100 compounds are depsidone [4]. In addition, in the
structure of this plant, certain chemical elements, organic
compounds, polysaccharides, carotenoids and other
substances have been identified [3, 4]. Lichen compounds
can be arbitrarily divided into two groups: primary and
secondary compounds. Primary lichen compounds have
structural functions and are involved in cell metabolism.
Secondary lichen compounds are characterized by acid
properties, such as lichen acids [5, 6]. Each species of
lichen has its own set of lichen acids, which generally give
qualitative reactions enabling the lichen species to be
discriminated. A lot of lichen species contain atranorin,
usnic acid, lecanoric acid, salazinic acid, lobar acid and
other acids [7]. Lichen acids are important not only to
identify the lichens, but also could be used as natural
antibiotics [8, 9]. Therefore, the bioactive compounds from
lichens have various biopharmaceutical applications as
antimicrobial, antioxidant and cytotoxic agents, being used
in the development of new formulations or technologies
for the benefit of human life [10].
Lichens are vegetable products with a significant
antioxidant [11], antimicrobial [12], anti-proliferative [13],
antifungal [14] and UV filter activity [15], belonging to the
Lichenophyta phylum, which is the least exploited
subdivision of fungus [16, 17]. In this article, chemical and
biological studies of the bioactive compounds from a native
species of lichens, Cetraria islandica, from the Cetraria
genus (Parmeliaceae family) [18] were carried out. The
* email: rodica.dinica@ugal.ro; Phone: 0745930740
2186 http://www.revistadechimie.ro
analyzed bioactive compounds are responsible for the
antioxidant and antimicrobial properties of this vegetable
and play an important role to treat various respiratory and
digestive diseases and to improve bronchial disorders [19,
20].
Experimental part
Plant material
In this study, local plant material represented by thallus
of Cetraria islandica lichen was used. The plant material
was harvested from the spontaneous flora of Fagaras
Mountains (Romania) in May 2015. Macroscopic analysis
of the plant product was done by inspecting the structural
appearance of erect lobes of the thallus, which were about
10 cm tall. To perform further analysis, the lobes were
divided in two, being flat and twist and with thick and rigid
cilia on the edge. The upper face of thallus was
characterized by a glossy greenish-brown color, while the
underside showed a white-gray, reddish color at the
insertion site of the substrate. Morphological characteristics
of the plant material were investigated by smell and taste
perception. Thus, the thallus of C. islandica lichen has bitter
taste and weak odor.
Extraction of furan derivatives from plant material
50 g samples of dry and ground plant material were
extracted with 250 mL acetone and 250 mL ethanol 95 %,
respectively under continuous stirring for 3 hours at 50°C.
The extracts were concentrated to 10 mL. The acetone
extract was precipitated with benzene to obtain usnic acid
as a white-yellow precipitate that was dried in the oven.
200 mg of usnic acid was obtained.
Infrared spectroscopy
The transmittance values in infrared were obtained in
range of 4000-400 cm-1 using Attenuated Total Reflectance
Fourier Transform Infrared Spectroscopy (ATR-FTIR) [21]
performed with Nicolet iS50 ATR equipment at room
temperature.
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Quantitative evaluation of usnic acid
The quantitative evaluation of usnic from acetone
extract of C. islandica lichen was perfomed by HPTLC
technique using Linomat IV equipment (Camag). The two
extracts of lichen and standard solution of usnic acid in
various concentrations were spotted on silica gel
chromatographic plate (60, F254) of 10 x 10 cm. The plate
was eluted in the chromatographic mobile phase, toluene:
dioxane: acetic acid (180:45:5) and dried for 5 minusing
TLC dryer from Linomat IV equipment. The qualitative
evaluation of colored bands was performed using Camag
TLC scanner II.
Antioxidant activity evaluation
The antioxidant activity of the extracts of C. islandica
lichen was determined using the radical scavenging
capacity assay, the reducing power of the extracts and the
hydrogen peroxide (H 2O2) scavenging capacity of the
extracts, respectively.
Reducing power assay
The reducing power of the two lichen extracts (acetone
and ethanol) was evaluated using the method of Yang,
Guo and Yuan [22], with some modifications. Rutin in
concentration of 0.1g/100mL was used as standard
according to the modified method. Samples were
measured spectrophotometrically at 420 nm. Each test
was performed in triplicate.
DPPH radical scavenging activity
The scavenging capacity assay was evaluated
according to the method of Bozin et al [23], with some
modifications [24, 25], using 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH). 1 mL of lichen extracts were mixed with
1 mL of 90 ìM DPPH, being brought with MeOH at final
volume of 4 mL. The same procedure was used to obtain
the blank test solution. The solutions were kept for 1 hour
at room temperature and the absorbance was recorded at
517 nm. Each test was performed in triplicate. The radical
scavenging capacity was calculated using the equation:
RSC (%) = 100 x (Ablank – Asample)/ Ablank
where, Ablank and Asample are the absorbance values of the
blank sample and of the test sample respectively, after 1
hour.
Hydrogen peroxide scavenging capacity assay
The hydrogen peroxide (H2O2) scavenging capacity of
lichen extracts was determined according to the method
of Ruch et al [26], with some modifications [24]. A solution
of H2O2 (2 mmol/L) was prepared in phosphate buffer (pH
7.4). To obtain the test sample, 0.6 mL of phosphate buffer
was added to 3.4 mL pondweed extracts. The absorbance
value of the reaction mixture was recorded at 230 nm.
Three replicates were made for each sample in
comparisons with rutin as standard solution. The
percentage of H2O2 scavenging capacity was calculated
as:
% scavenged H2O2 = [(A0- A1) /A0] x 100 (2)
where, A0 is the absorbance of the control and A1 is the
absorbance in the presence of the sample or standards.
Antimicrobial activity evaluation
To test the sensitivity/resistance of various
microorganisms to lichen extracts, the diffusion method
(Kirby-Bauer) [27, 28] and agar dilution method [29] were
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used. All strains were obtained from the Clinic Laboratory
of Faculty of Medicine and Pharmacy, Dunarea de Jos
University of Galati: the G(-) Gram-negative bacteria
Enterobacter hormaechei (ATCC 700323), Escherichia coli
(ATCC 25922), Proteus mirabilis (ATCC 29906),
Pseudomonas aeruginosa (ATCC 211853), Salmonella
enteritidis (ATCC 13076); G(+) Gram-positive bacteria
S taphylococcus aureus (ATCC 25293), Streptococcus
pyogenes (ATCC 19615), Enterococcus casseliflavus (ATCC
25788) and fungus Candida albicans (ATCC 10259).
For each of the strains, the diffusion method was
performed, in order to assess the sensitivity of the strains
to acetone and alcoholic extracts, usnic acid dissolved in
dimethyl sulfoxide (DMSO), antibiotics (gentamycin and
pencillin) in case of bacteria and antifungal (nystatin) for
fungi. The culture media used were: Müller Hinton (Gram-
negative bacteria, Staphylococcus aureus), blood agar
(Enterococcus casseliflavus, Streptococcus pyogenes) and
solid Sabouraud medium for fungus. This method consists
of the following steps: (i) inoculum preparation; (ii)
inoculum seeding; (iii) blank disc soaking; (iv) micro tables
and discs deposition; (v) incubating; (vi) reading. For each
bacterial strain isolated in pure culture, an inoculum was
prepared with the turbidity of 0.5 McFarland (1.5x108 CFU/
mL), the nephelometry being controled with Densi CHEK
apparatus. Five identical colonies suspended in 3 mL saline
solution for bacteria/fungus were used. The seeding was
performed uniformly on the media surface. After seeding,
plates were incubated for 5-10 minutes for the inoculum
to be absorbed into the medium. Meanwhile the blank discs
were soaked with 25 µl ethanol extract, acetone extract
and usnic acid solution, respectively. The soaked disks and
micro tablets of gentamycin, pencillin and nystatin of
known concentrations (10 µg, 100 U.I.) were applied using
a forceps by pressing gently on the surface of the seeded
medium. In their application, a distance of ~15mm from
the edge of the plate and ~30mm between micro tables/
discs was developed. After 10-15 min of application, the
plates were incubated aerobically at 37oC for 24 h. The
antimicrobial compound impregnated on the disc diffuses
in the medium and shows an inhibition zone of the culture
(1) around the disc, depending on its sensitivity. After
incubation, only the plates with an increase in terms of
culture purity and density were evaluated. The inhibition
zone was measured 2-3 times in different directions, with
a ruler. For transparent medium, the assessment was done
directly on the bottom plate and in case of blood agar plates
the measurement of inhibition zone diameter was
obtained at the medium surface.
The dilution method was used to determine the
sensitivity of the microorganism from the test sample
indicating the concentration required to inhibit its
development. In this way the minimum inhibitory
concentration (MIC) was measured. Increasing dilutions
of usnic acid dissolved in DMSO were prepared directly in
saline solution. Equal amounts of the analyzed culture were
seeded to determine the maximum dilution of the
substance which inhibits the strain. The stock solution was
prepared from usnic acid dissolved in DMSO whose initial
concentration was 50 mg/mL. Pseudomonas aeruginosa
was the evaluated microorganism. An inoculum with the
turbidity of 0.5 McFarland (1.5x10 8 CFU/mL) was
performed and controlled nephelometry using Densi CHEK
apparatus. Five identical colonies were suspended in about
3 mL of saline solution to test the antimicrobial activity of
bacteria. Equal amounts from the final solution were
seeded into all dilutions of the usnic acid. For dilutions 10
test tubes were used and filled with 450 mL of saline
solution, adding 2.50 µL of usnic acid stock solution. After
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homogenization, 50 µL were passed in the next test tube
and so on until the tube 10 from which 50 µL of solution
were removed. The quantification was done after
incubation at 37oC for 24 h. The minimum inhibitory
concentration (MIC) was measured by visual appreciation
of the tube in which no bacterial growth was identified
(the culture medium was clear). To appreciate the
minimum bactericidal concentration (MBC), by observing
the absence of bacterial growth, the inoculum was seeded
from tubes 3 to 10 into Petri plates using Müller Hinton
medium.
Results and discussions
Chemical analysis of furan compounds
Qualitative and quantitative evaluation of usnic acid from
acetone extract was performed using IR spectroscopy and
HPTLC technique.
IR analysis
FTIR spectra recorded for the acetone extract is shown
in figure 1. From this spectrum, a band at 1690 cm-1
corresponding to a conjugated cyclic ketone groups (C =
O) was identified. Also, some weak bands at 1715 and
1678 cm-1 corresponding to the non-conjugated cyclic
methyl ketones groups were detected. Characteristic
signals of aryl alkyl ether group (C-O-C) were recorded at
1283 and 1072 cm-1.
Fig.1. IR spectrum of acetone extract
HPTLC analysis
The presence of usnic acid in the structure of C. islandica
was identified using higher pressure thin layer
chromatography. The colored spots from the chromato-
graphic plate with the Rf corresponding to usnic acid were
detected at a wavelength of 237 nm (fig. 2).
Fig. 2. Chromatogram of lichen extracts (acetone extract - P1 and
ethanol extract – P2) and the standard solution of usnic acid (P3)
Rf value identified for the usnic acid (0.18) was used in
the quantitative assessment of usnic acid by quantification
of the usnic acid concentration in the acetone and ethanol
extracts. The usnic acid concentration was measured by
plotting the calibration curve of the standard solution of
usnic acid (fig. 3).
2188 http://www.revistadechimie.ro
Fig. 3. Calibration curve of usnic acid (R = 0.9214, sdv = 11.5 %)
The usnic acid concentration from the ethanol extract
(4.782 µg/µL) and acetone extract (5.600 µg/µL),
respectively was obtained by interpolation of this calibration
curve. The highest concentration of usnic acid was found
for acetone extract.
Biological activity of furan derivatives from C. islandica
lichen
Antioxidant activity
The determination of reducing power serves as an
important indicator regarding to a potential antioxidant
activity of the compound or mixture studied. Many studies
have revealed that there is a direct correlation between
the antioxidant activity and reducing power of plant
components [30]. To measure the reducing power, the
transformation of Fe3+ to Fe2+ in presence of the acetone
and ethanol extracts was investigated using the method
described by Yang, Guo and Yuan (2008), with some
changes. The reducing capacity of samples in comparison
with rutin solution, known for its notable antioxidant activity
is shown in figure 4. A high absorbance of the reaction
mixture indicates a higher reducing power.
Fig. 4. Reducing power evaluation of Cetraria islandica extracts
This result suggests that C. islandica extracts have a
remarkable potential to donate electrons to reactive free
radicals, making them more stable species, non-reactive
and ending the chain radical reaction.
The results expressed by percentage of DPPH
scavenging are shown in figure 5 and varies thus: 18.62 %
for ethanol extract and 40.68 % for acetone extract.
The scavenging capacity varies for the analyzed extracts
and the results are presented in figure 6.
Our results have shown that the percentage inhibition
capacity ranged from 57.02 % (ethanol extract) to 63.16 %
(acetone extract). Therefore, the acetone extract, that was
shown to have a greater concentration of usnic acid, also
REV.CHIM.(Bucharest)♦70♦ No. 6 ♦ 2019
 Fig. 5. DPPH evaluation of Cetraria islandica extracts
Fig. 6. Scavenging activity evaluation of Cetraria islandica extracts
exhibited higher reducing power and higher radical
scavenging capacity than the ethanol extract.
Antimicrobial activity
The antimicrobial activity of C. islandica extracts was
assessed using the diffusion method (Kirby-Bauer) and the
dilution method. The diffusion method provides information
on the resistance of plant material extracts against various
microorganisms [31]. In this method, the following areas
were evaluated: (i) inhibition zone diameters (area without
microbial colonies, including the diameter of disc with
antibiotic) (fig. 7a) and (ii) well-developed colonies
appeared within of the inhibition zone (fig. 7b).
a b
Fig. 7. (a). Sensitivity of Enterococcus casselifavus against lichen
extracts ; (b). Sensitivity of Pseudomonas aeruginosa against lichen
extracts
Lichen extracts and usnic acid solution dissolved in
dimethyl sulfoxide showed a significant inhibition activity
on Gram-negative bacteria (fig. 8). So, the extracts and
usnic acid have antibacterial activity on most Gram-
negative strains considered in this study.
The acetone extract showed a greater antimicrobial
activity on Enterobacter hormaechei bacteria compared
with penicillin. For Proteus spp. bacteria, the ethanol extract
was characterized by a superior antimicrobial activity
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Fig. 8. Antimicrobial activity of the lichen extracts and usnic acid
compared with the antibiotic on Gram-negative bacteria
compared to usnic acid and acetone extract, indicating
that other bioactive compounds are responsible for this
behavior. Except for the antibiotic, the usnic acid dissolved
in dimethyl sulfoxide was the only tested compound which
had antibacterial activity on Salmonella enteridis bacteria.
The antimicrobial activity of C. islandica extracts and
usnic acid against some Gram-positive bacteria was
evaluated by measuring the diameters of the inhibition
zones around of the disc in mm (fig. 9).
Fig. 9. Antimicrobial activity of Cetraria islandica extracts against
Gram positive bacteria
Inhibition zones of the growth of Gram-positive bacteria
were increased both for lichen extracts and for usnic acid
for a content of 25 µL/blank disc. For Streptococcus
pyogenes bacteria the results are different for each extract
and antibiotic used. The ethanol extract has the highest
antimicrobial potential (diameter of the inhibition zone of
27 mm) compared to usnic acid (diameter of the inhibition
zone of 25 mm), the acetone extract (diameter of the
inhibition zone of 22 mm) and pencillin (diameter of the
inhibition zone of 18 mm). The antimicrobial potential of
the ethanol extract was close to the one of pencillin. The
acetone extracts showed an antibacterial activity identical
to that of the ethanol extract against of the strain of the
Staphylococcus aureus bacteria and Enterococcus
casselifavus bacteria, respectively.
Dulger et al. (1998) investigated ethyl acetate, acetone,
chloroform and ethanol extracts of C. islandica against
different microorganisms by disc-diffusion method. They
have found that C. islandica exhibited antimicrobial activity
against some Gram-positive bacteria, but had no
antimicrobial activity against Gram-negative bacteria and
fungi [32]. Differences in the results could be explained by
the difference in sensitivity of tested microorganisms,
different methods of testing and the solvents used. These
results could be expected due to the fact that numerous
2189
tests have proven that bacteria are more sensitive to
antibiotics compared to fungi [33].
The antifungal activity of the lichen extracts and usnic
acid against Candida albicans strain were assessed in
comparison with the Nystatin activity (fig. 10).
Fig. 10. Antifungal activity of lichen extracts against Candida
albicans strain
By measuring the diameters of the inhibition zones
around the disc, expressed in mm, it was observed that
the acetone extract (inhibition diameter of 20 mm) has an
antifungal activity against Candida albicans strain similar
to that of Nystatin (inhibition diameter of 24 mm), an
elective fungal, followed by usnic acid (inhibition diameter
of 16 mm) .
The reason for different sensitivity between fungi and
bacteria could also be found in the difference in
permeability of the cell wall. The cell walls of Gram-positive
bacteria consist of peptidoglycan (murein) and teichoic
acids, while the cell walls of Gram-negative bacteria consist
of lipopolysaccharides and lipopoliproteins [34]. On the
other hand, the cell walls of fungi consist of poly-
saccharides such as chitin and glucan [35].
Antimicrobial activity using dilution method
The minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) of usnic acid
against Pseudomonas aeruginosa strain were measured
using dilution method (fig. 11). The dilutions (from the
stock solution) were prepared from 1:2 to 0.048:50 mg/
mL.
Fig. 11. Minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) determination. (concentration of
usnic acid stock solution: 50 mg/mL).
It was observed that usnic acid has antimicrobial activity
against Pseudomonas aeruginosa, the MIC being 6250 µg/
mL. The minimum bactericidal concentration (3125 µg/
mL) was appreciated by observing the absence of colonies
growth seeded on Petri plates with Müller-Hinton medium
from analysed samples.
Different lichens were screened for antimicrobial activity
in search of new antimicrobial agents. Kosaniç et al.
(2012a) found antimicrobial activity for the acetone extract
of the lichens Umbilicaria crustulosa, U. cylindrica, and U.
polyphylla [36]. Similar results were reported by Karthikai
Devi et al. (2011) for different extracts extracted from the
lichen Roccella belangeriana [37]. Goel et al. (2011) found
2190 http://www.revistadechimie.ro
that the lichen Parmotrema reticulatum had a strong
antimicrobial effect [38].
Conclusions
Lichens are plants that contain in their structure many
constituents and are therefore very capable of variation.
So, it is very important to know the characteristic chemical
components and the pharmacologically active compounds
of the lichens.
The main goal of this study was to identify and extract
the bioactive compounds with antioxidant and anti-
microbial potential from C. islandica lichen.
The acetone and ethanol extracts were obtained using
solid-liquid extraction method and the usnic acid was
extracted from acetone extract by precipitation with
benzene. Extract samples confirmed the presence of
carbonyl compounds, usnic and salazinic acid in their
composition, according to literature [39, 40].
The HPTLC analysis was able to provide useful
qualitative and quantitative data for the quick determination
of the usnic acid in the acetonic and ethanolic extract,
therefore this technique being ideally suited for the analysis
of botanical products.
Both acetone and ethanol extract showed a superior
antioxidant activity compared to other standard
compounds used to evaluate the antioxidant activity.
The assessment of the antimicrobial activity of C.
islandica extracts by diffusion method (Kirby-Bauer) and
dilution method highlighted that both extracts and pure
usnic acid have activity against certain Gram-positive and
Gram-negative bacteria and fungi such as Candida
albicans. Antimicrobial activity depends on the solvent and
microbial strain used. The acetone extract has a better
antimicrobial activity compared to penicillin against
Streptococcus pyogenes bacteria.
The lichen extracts, according to the technology adopted,
could be used as the basis of some pharmaceutical
formulations due to pharmacological potential of biological
active compounds from C. islandica species.
Acknowledgements: : This work was supported by a grant of the
Romanian National Authority for Scientific Research, CNCS – UEFISCDI,
project number PN-II-ID-PCE-2011-3- 0226 and CENTRUL ROMÂN
PENTRU MODEL AREA SISTEMELOR RECIRCUL ANTE DE
ACVACULTURÃ, Axa 2 POSCCE, operaþiunea O2.2.1 - Dezvoltarea
infrastructurii C-D existente ºi crearea de noi infrastructuri C-D, 2014-
2015.
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